P964: IMMUNOPHENOTYPED-SUSPENSION-MULTIPLEX FISH BY IMAGING FLOW CYTOMETRY FOR THE SIMULTANEOUS DIAGNOSIS OF THREE PIVOTAL IGH TRANSLOCATIONS IN MULTIPLE MYELOMA
Authors: T. Tsukamoto, M. Kinoshita, K. Yamada, T. Yamaguchi, Y. Chinen, S. Mizutani, T. Fujino, Y. Shimura, T. Kobayashi, J. Inazawa, J. Kuroda
Journal: HemaSphere (2022)

Background: The cytogenetic abnormalities including t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32) are associated with the molecular profiles, clinical features, and treatment outcome in multiple myeloma (MM). Although the double-color interphase fluorescence in situ hybridization (DC-FISH) is well established to detect those translocations, the conventional method can analyze only one abnormality on each slide. Aims: To analyze multiple chromosomal abnormalities simultaneously with high sensitivity and specificity, we aimed to develop a new diagnostic modality of four-color FISH for multiple translocations in immunophenotyped cells in suspension using imaging flow cytometry (immunophenotyped-suspension-multiplex (ISM)-FISH). Methods: For the ISM-FISH, we first subjected Carnoy-fixed cells in suspension to immunostaining using a brilliant violet 421-conjugated anti-CD138 antibody. Then, we hybridized with four different FISH probes, i.e., Texas Red (TxRed)-conjugated probe for IGH, fluorescein isothiocyanate (FITC)-conjugated probe for FGFR3, Gold-conjugated probe for cyclin D1 (CCND1), and Cy5-conjugated probe for c-MAF, in suspension. Then, more than 2.5 × 104 nucleated cells for each sample were analyzed with imaging flow cytometer MI-1000 (Sysmex) by six channels: one for immunophenotyping, four for FISH and one for bright field image. In the process of imaging flowcytometric analysis, we sorted CD138-positive cell fraction, computed the distance between FISH probes using originally developed spot counting tool, and determined to be positive for translocation when the distance is under the threshold. Results: By this ISM-FISH system, we simultaneously examined three chromosomal translocations, including t(4;14), t(14;16), and t(11;14), in three MM cell lines, KMS-11, KMS-21-BM, KMS-26, and successfully detect the abnormalities. Using different proportions of transformation-positive MM cells spiked with transformation-negative leukemic cells, HL-60, ISM-FISH showed a sensitivity of less than 0.1%. Next, ISM-FISH and conventional DC-FISH were performed for bone marrow nucleated cells from 70 patients with MM or monoclonal gammopathy of undetermined significance (MGUS) and the experiments showed a promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive than standard DC-FISH, examining 200 interphase cells with its optimal sensitivity of up to 1.0%. Furthermore, the ISM-FISH showed a positive concordance of 83.3% and negative concordance of 97.4% using standard DC-FISH, examining 200 interphase cells. Furthermore, in three patients (4%), t(4;14) or t(11;14) were positive by ISM-FISH but negative by DC-FISH examining 200 cells. Extensive DC-FISH study examining additional 1,000 cells showed positive in these ISM-FISH-positive/DC-FISH (200cells)-negative samples, suggesting higher sensitivity of ISM-FISH than that of the standard DC-FISH. Image:Summary/Conclusion: This study developed the new diagnostic system of ISM-FISH, which enables the simultaneous diagnosis of three clinically pivotal chromosomal translocations, t(4;14), t(14;16), and t(11;14), in MGUS and MM. This system may facilitate rapid and reliable cytogenetic diagnosis and promote patient-oriented therapy according to the type of chromosomal translocation in the clinical practice setting.